**3.2.1.3 Analytical methods**

204 Biogas

for inoculation were prepared by growing the yeast in a 1 L baffled shake-flask containing sterile water and 100 mL YPG medium at 30 ºC for 24 h on orbital shaker table at 200 rpm to a concentration of approximately 108 cells mL-1. The cultures in baffled shaken flasks of 100 mL were used to prepare the inocula. After 24 h of incubation at 30 ºC, the precultures were centrifuged at 3800 rpm for 10 min and the cells were resuspended in sterile water to obtain 106 cells mL-1. Enzyme β-D-galactosidase (optimum temperature 30 ºC, optimum acidity pH 4.5-5.0, activity 8.7 AU mg-1 d.m. of the preparation), from *Aspergillus oryzae* manufactured by the SIGMA company (USA), was used for co-immobilization process. The amount of yeast and enzyme was 3% free cell inoculum and 4 cm3 enzyme solution. The yeast culture was co-immobilized in the 2% (w/v) sodium alginate by dropping yeast and enzyme into 150 cm3 0.09 mol L-1 solution of CaCl2 with 10% glucose. The cell beads were washed with sterile water and were stored as a fermentation medium in physiological solution at 8C.

UF whey permeate (non-deproteinized, non diluted and non-sterilized) with the average lactose concentration of 50 g L-1 from the Dairy Plant in Nowy Dwór Gdański, Poland, was

Continuous fermentation was carried out in the laboratory-scale plant consisted of the two UASB reactors with a working volume of 5 L each (Fig. 8). These two reactors were used to enable parallel test series with and without ultrasound irradiation. The fermentation medium was pumped continuously to the bottom part of the reaction tank by means of the peristaltic pumps. The necessary mixing was achieved through the upward wastewater flow. The reactors were water-jacketed and operated at a constant temperature of 30±1 ºC. The pH of mixed liquid in the reactors was controlled automatically at pH 5.1 ± 0.2 with 2 M

The reactors were inoculated with 40% (v/v) solid beads containing the immobilized cells which corresponded to 39.4 g cells dry weight - DW L-1 of working bioreactor volume. After adding the cell beads inoculum to the bioreactors, before starting continuous feeding, a batch fermentation was conducted for 24 h under additional gentle agitation (100 rpm). Next the reactors worked at different HRTs of 12, 24 and 36 h. At each HRT the reactor was operated till it has reached the steady-state (the steady-state conditions were evidenced

**3.2.1.2 Fermentation medium and experimental system** 

used as a fermentation substrate (Table 2).

Fig. 8. A scheme of the research station.

NaOH.

Lactose and ethanol concentrations in the effluent distillate were determined according to Standard Methods (PN-67/A-86430; PN-A-79528-3:2007). The samples were analyzed in triplicates and results were reproducible within 3% deviation.

All fermentation steps connected with different HRTs were carried out in triplicate. Significant differences between the effects obtained in the two reactors with and without ultrasound exposure were analysed using an ANOVA *F*-test (Statistica 7.1 software, Statsoft Inc.) A 5% probability level was applied for all the tests. If p<0.05 from an ANOVA *F*-test, the differences between the effects were considered to be significantly different from one another.
